Protein secondary structure in spider silk nanofibrils

Nanofibrils play a pivotal role in spider silk and are responsible for many of the impressive properties of this unique natural material. However, little is known about the internal structure of these protein fibrils. We carry out polarized Raman and polarized Fourier-transform infrared spectroscopies on native spider silk nanofibrils and determine the concentrations of six distinct protein secondary structures, including β-sheets, and two types of helical structures, for which we also determine orientation distributions. Our advancements in peak assignments are in full agreement with the published silk vibrational spectroscopy literature. We further corroborate our findings with X-ray diffraction and magic-angle spinning nuclear magnetic resonance experiments. Based on the latter and on polypeptide Raman spectra, we assess the role of key amino acids in different secondary structures. For the recluse spider we develop a highly detailed structural model, featuring seven levels of structural hierarchy. The approaches we develop are directly applicable to other proteinaceous materials.


Raman spectra with different polarizations
Supplementary Figure  (a) XX direction; (b) ZZ direction; (c) YY direction; (d) ZX direction; (e) XZ direction. The YY and XX spectra are similar across the measured wavenumber range, where the YY spectrum features a lower intensity. ZX and XZ spectra are nearly identical. Thick gray lines: experimentally (Exp.) measured spectra. Dashed black line: multi-peak fit; all constituting sub-peaks shown in colors. Blue, purple, red, and yellow peaks are assigned to β-sheet, β-turn, helical, and random coil peaks, repsectively. Green peaks represent amino acid residues with aromatic side-chain groups. Gray peaks are unassigned. The colored horizontal bars represent known peaks from the literature, with colors matching the secondary structure assignment.

Wide range FTIR and Raman spectra
Supplementary Figure 3. Unpolarized FTIR and Raman spectra.
(a) Unpolarized, multi-strand FTIR spectrum and (b) single-strand Raman spectrum of Loxosceles silk over 500-4000 cm -1 . Amide-I/II/III, A, and B bands and some major peaks in 2800-3100 cm -1 are labeled with peak positions.
Supplementary Figure 3 features FTIR and Raman spectra of Loxosceles silk ribbon in the 500-4000 cm −1 range. Two distinct groups of peaks, one from ≈700-1700 cm −1 and one from ≈2800-3300 cm −1 , can be identified in these wide-range spectra. It is clear that the relative intensities among amide bands are significantly different between FTIR and Raman results.

Error estimation of P ZX values
In order to estimate the standard deviation of the Raman P ZX values featured in Figure 4 in the main text, we introduced uniformly distributed errors to the initial fitting parameters for the sub-peaks. The maximum error caused by the noise generated for the peak position, FWHM, peak magnitude, and Lorentzian content were limited to ± 5 cm −1 , ± 5cm −1 , 10%, and 0%-100%, respectively. Multiple optimization tests (n = 15) have been made to fit the experimental Raman spectra. The error bars in Figure 4a represent the standard deviation for each Raman P ZX value.
In order to estimate the standard deviation associated with the absorbance subpeaks in the p-FTIR data, we minimized the mean squared error (MSE) between the model and experimental data using the Levenberg-Marquardt algorithm. The process was done using the proprietary package WVASE32. The amplitude and bandwidth of the oscillators in the dielectric function were varied to produce a minimum in the MSE. The error bound on these fits was calculated by evaluating the standard 90% confidence limit (SCL). The uncertainty in the P values can be calculated by propagating the uncertainty: Using the form | | | | | | | | above in equation (1), we obtained the uncertainty in the P values and presented them in Figure 4a in the main text as error bars.

Orientation distribution of different secondary structures
Based on the symmetry of the silk ribbon and on the knowledge of the natural spinning process of a silk fiber, we assume that any orientation mechanism generates preferred alignment relative to one of the Cartesian axes (X, Y, or Z). Imperfect orientation, thus leads to a distribution of angles around this Cartesian axis, which we assume to be Gaussian (N( )).
Based on our polarization characterization in a given plane (ZX, ZY, or XY), we can then estimate the standard deviation σ of this Gaussian distribution from the corresponding P value in this plane. For example, in the ZX plane: where A and θ are the dipole moment/polarizability A and its angle θ from the axis. Since , we can numerically find the σ that gives the experimentally observed P ZX . From the expression of N( ), we can derive that the half width at half maximum (HWHM) is √ . For positive P ZX (preferred alignment with the Z axis), this distribution is around the Z axis; for negative P ZX , this angular distribution is relative to the X axis. According to our calculations, HWHM becomes >90º for P<0.0284 (very weak orientation preference). The HWHM values for the secondary structure related peaks calculated from p-Raman and p-FTIR spectra are featured in Supplementary Tables 2 and 3.  Table 3. Half width at half maximum (HWHM) values for the secondary structure orientation distribution in the ZX plane, calculated from p-FTIR spectra. Abbrivations: s, stretching; b, bending; ib: in-plane bending; sb, symmetric bending; ab, antisymmetric bending.

Transition dipole coupling (TDC) in antiparallel (Ala) n -sheet
In the amide-I band, the FTIR peaks at 1697 and 1631 cm −1 demonstrated inverse dichroism (Figures 2a and 2b of the main text). It has been proposed that they are two split peaks caused by transition dipole coupling (TDC) between neighboring C=O groups on adjacent peptide strands in antiparallel -sheets. [1][2][3] The associated inverse dichroism can be explained with their corresponding vibration modes (Supplementary Figures 4a-d,

Intrinsic P ZX value estimation for 3 1 -and -helices
To estimate the intrinsic P ZX values of 3 1 -and -helices, we calculated the oscillator intensities along Z' (helix axial direction) and X (direction perpendicular to helix axis) directions for different groups, based on previously published atom coordinates. For the 3 1 -helices, we employed the coordinates given by Ramachandran et al. 5 For -helices, we used atom coordinates of the -helix segment (KANADAFINSFISAAS) of a MaSpI N-terminal domain model 2N3E. 6 The estimated intrinsic P zx values are presented in Supplementary

X-ray diffraction measurement of Loxosceles silk
To prepare the sample, approximately 40 mg of Loxosceles silk collected from both male and female spiders was first pressed into a small thin disk (1 cm diameter, 2 mm thickness). Subsequent X-ray diffraction experiment on the sample was performed on a Bruker APEX DUO diffractometer equipped with an APEX II CCD detector and a microfocus copper K α source (wavelength = 1.54 Å). The sharp and broad peaks are assigned to crystalline structure and amorphous structure, respectively. The ratio between the sum of crystalline peak magnitudes and the sum of amorphous peak magnitudes is used to represent the crystalline percentage, 43.2%.

Calculated unpolarized FTIR spectrum
Supplementary Figure 6. Calculated unpolarized FTIR spectrum and its sub-peak decomposition of the amide-I band.
The -sheet content is determined to be 41±5% from the amide-I band. Black solid line: experimentally measured spectrum; red dashed line: multi-peak fit; yellow solid line: deconvoluted peaks for -sheet strcture; blue solid line: deconvoluted peak for -helix strcture.
In order to calculate the β-sheet composition from the FTIR spectra, we used the pseudounpolarized spectrum (Supplementary Figure 6). It is necessary to use pseudo-unpolarized data, since the secondary structures are not along only one direction but are instead distributed at different orientations in the plane of the silk. If polarized data was to be used, a different volumetric percentage would be calculated for different polarization directions. We removed the directional dependence of the secondary structures by using the pseudo-unpolarized spectrum, which has no directional dependence. The Loxosceles spider silk ribbons are only 50 nm thin in Y direction, orders of magnitude less than in the X and Z directions, and thus, they can be approximated as a quasi-two-dimensional material. Consequently, the Y direction was not considered, and the pseudo-unpolarized spectrum was calculated by averaging the transmission spectra of the parallel (Z) and perpendicular (X) data sets, (|Z|+|X|)/2. The β-sheet percentage calculated represents the average β-sheet composition in the ZX plane of the silk. The pseudounpolarized absorbance spectrum was deconvolved, using the same methods as described previously for the p-FTIR data sets. The β-sheet composition was calculated for the amide-I band, by taking the ratio between the area of the β-sheet oscillators to the total area of the amide-I band absorbance. This method of secondary structural composition calculation has often been used in the literature. [8][9][10][11] FTIR spectroscopy on samples with a different geometry may require different methods of calculating pseudo-unpolarized spectra. For instance, for cylindrical fibers, it has been shown that a uniaxial symmetry, and thus |X|=|Y|, applies, which leads to (|Z|+2|X|)/3 for unpolarized FTIR spectra. 13

Calculated unpolarized Raman spectrum
Calculation of the pseudo-unpolarized spectra is somewhat more complicated for Raman data than for the FTIR spectra discussed in section 10, because there can be up to 16 unknown variables. A mathematically rigorous solution for this problem is challenging and has yet to be demonstrated. We developed the following approximation based on prior work. 12,13 Since we experimentally obtained Raman spectra with the polarizations XX, YY, ZZ, XZ, and ZX, and then found the YY and XX spectra to be similar and with an intensity ratio of 2/3, we assumed

Proposed components in mixed peaks
In Supplementary Table 5, we summarize the potential components in the peaks with more than one contributing secondary structure (peaks labeled with dashed multi-color stripes in Figure 2 in the main text).

Ratio of β-sheets and helices based on amide-III analysis
Based on Raman amide-I analysis, the ratio of β-sheets vs helices is 44%/15.9% ≈ 2.8.
We looked at the independent Raman amide-III peaks and considered the intensity ratios of three peaks: the β-sheet peak at 1224 cm −1 , the helical peak at 1266 cm −1 , and the 1241 cm −1 peak, which has been proposed to have both helical and β-sheet contributions. The ZZ area percentages and P ZX values of these three peaks are {0.65, 0.16, 0.19} and {0.64, 0.36, 0.39}, respectively.
The helical component of the last peak is a low-frequency shoulder to the 1266 cm −1 peak; its βsheet component is a high-frequency shoulder to the 1224 cm −1 peak. By considering its P ZX values of the three peaks, we estimated that the 1241 cm −1 peak is 89% due to helical structures and 11% caused by β-sheets. Using this to calculate the β-sheet vs. helix ratio for all three amide-III peaks, we found 2.0. The same calculation can be carried out for the 3 amide-III FTIR peaks at 1218 cm −1 (β-sheet), 1236 cm −1 (mixed β-sheet and random coil), and 1265 cm −1 (helical), yielding a 55% β-sheet and 45% random coil contributions for the mixed peak. The corresponding β-sheet vs. helix ratio was similar, 1.7.